Recombinant genes for producing proteins in plants comprise in sequence the following operably linked elements: a promoter which functions in plants, a structural gene encoding the target protein, and a non-translated region which also functions in plants to cause the addition of polyadenylated nucleotides to the RNA sequence. Much scientific effort has been directed to the improvement of these recombinant plant genes in order to achieve the expression of larger amounts of the target protein.
One advantage of higher levels of expression is that fewer numbers of transgenic plants would need to be produced and screened in order to recover plants which produce agronomically significant quantities of the target protein. High level expression of the target protein often leads to plants which exhibit commercially important properties.
Improved recombinant plant genes have been generated by using stronger promoters, such as promoters from plant viruses. Further improvements in expression have been obtained in gene constructs by placing enhancer sequences 5' to the promoter. Still further improvements have been achieved, especially in monocot plants, by gene constructs which have introns in the non-translated leader positioned between the promoter and the structural gene coding sequence. For example, Callis et al. (1987) Genes and Development, Vol. 1, pp. 1183-1200, reported that the presence of alcohol dehydrogenase-1 (Adh-1) introns or Bronze-1 introns resulted in higher levels of expression. Dietrich et al. (1987) reported that the length of the 5' non-translated leader was important for gene expression in protoplasts. Mascarenkas et al. (1990) reported a 12-fold and 20-fold enhancement of CAT expression by use of the Adh-1 intron.
Expression of recombinant plant genes may also be improved by the optimization of the non-translated leader sequences. These leader sequences are by definition located at the 5' end of the mRNA and are untranslated. The leader sequence is further defined as that portion of the mRNA molecule which extends from the 5' CAP site to the AUG protein translation initiation codon. This region of the mRNA plays a critical role in translation initiation and in the regulation of gene expression. For most eukaryotic mRNAs, translation initiates with the binding of the CAP binding protein to the mRNA cap. This is then followed by the binding of several other translation factors, as well as the 43S ribosome pre-initiation complex. This complex travels down the mRNA molecule while scanning for a AUG initiation codon in an appropriate sequence context. Once this has been found and with the addition of the 60S ribosomal subunit, the complete 80S initiation complex initiates protein translation (Pain 1986; Moldave 1985; Kozak 1986). A second class of mRNAs have been identified which possess translation initiation features different from those described above. Translation from these mRNAs initiates in a CAP-independent manner and is believed to initiate with the ribosome binding to internal portions of the leader sequence (Sonenberg 1990; Carrington and Freed 1990; Jackson et al. 1990).
The efficiency of translation initiation is determined by features of the 5' mRNA leader sequence, and presumably this ultimately affects the levels of gene expression. By optimizing the leader sequence, levels of gene expression can be maximized. In plant cells most studies have investigated the use of plant virus leaders for their effects on plant gene expression (Gallie et al. 1987; Jobling and Gehrke 1987; Skuzeski et al. 1990). The most significant increases in gene expression have been reported using the Tobacco Mosaic Virus Omega (TMV) leader sequence. When compared with other viral leader sequences, such as the Alfalfa Mosaic Virus RNA 4 (AMV) leader, two to three fold improvements in the levels of gene expression have been observed using the TMV Omega leader sequence (Gallie et al. 1987; Skuzeski et al. 1990). Larger increases in gene expression have been observed when comparisons were made with an artificial non-native leader sequence. No consensus regulatory sequences have been identified within the TMV leader sequence.
Like the TMV leader sequence, most 5' untranslated leader sequences are very A,U rich and are predicted to lack any significant secondary structure. One of the early steps in translation initiation is the relaxing or unwinding of the secondary. mRNA structure (Sonenberg 1990). Messenger RNA leader sequences with negligible secondary structure may not require this additional unwinding step and may therefore be more accessible to the translation initiation components. Introducing sequences which can form stable secondary structures reduces the level of gene expression (Kozak 1988; Pelletier and Sonenberg 1985). The ability of a leader sequence to interact with translational components may play a key role in affecting levels of subsequent gene expression.
In the search for leader sequences with improved properties, genes coding for heat shock proteins were scrutinized. Regulation of heat shock genes has been shown to occur at the transcriptional and translational level (Baumann et al. 1987; Kimpel and Key, 1985). Heat shock genes may be induced and expressed in response to hyperthermic stress (Key et al. 1981), as well as in response to other environmental conditions. During heat shock there is preferential translation of heat shock mRNAs (Storti et al. 1980). The translational control has been shown to be determined by the 5' untranslated leader sequence (McGarry and Lindquist 1985). A heat shock mRNA leader sequence operably linked to the mRNA of a non-heat shock gene would facilitate translation during heat shock conditions (Klemenz et al. 1985). The specific aspects of this regulation are not known. The heat shock mRNA 5' leader sequence may be more efficient at initiating translation, or may contain a particular structural feature that allows preferential translation during heat shock. Whatever the mechanism, the characteristics of the heat shock mRNA leader sequence may also provide an improvement to gene expression during non-heat shock conditions.
This invention makes a significant contribution to the art by providing non-translated leader sequences for use in genetic constructs which enhance gene expression in plants. The 5' non-translated leader sequences described herein provide for a significant increase in expression over other non-translated leader sequences which have been previously employed by those skilled in the art.